The invention generally relates to a device and method for photoactivating new and known compounds.
Whole blood collected from volunteer donors for transfusion recipients is typically separated into its components: red blood cells, platelets, and plasma. Each of these fractions is individually stored and used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia; the concentrated platelet component is used to control bleeding; and the plasma component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.
Ideally, all blood cell preparations should be from freshly drawn blood and then immediately transfused to the recipient. However, the logistics of operating a blood donor center preclude this possibility in the vast majority of cases. Transfusions are needed day and night and it is difficult, if not impossible, to arrange for donor recruiting at unusual hours. Consequently, modern blood donor centers must use stored blood products.
In the United States, blood storage procedures are subject to regulation by the government. The maximum storage periods for the blood components collected in these systems are specifically prescribed. For example, whole blood components collected in an xe2x80x9copenxe2x80x9d (i.e. non-sterile) system must, under governmental rules, be transfused within twenty-four hours and in most cases within six to eight hours. By contrast, when whole blood components are collected in a xe2x80x9cclosedxe2x80x9d (i.e. sterile) system the red blood cells can be stored up to forty-two days (depending upon the type of anticoagulant and storage medium used) and plasma may be frozen and stored for even longer periods.
Murphy and Gardner, New Eng.J.Med. 280:1094 (1969), demonstrated that platelets stored as platelet-rich plasma (PRP) at 22xc2x0 C. possessed a better in vivo half-life than those stored at 4xc2x0 C. Thus, more acceptable platelet concentrates could be transfused after storage at room temperature. Until recently, the rules allowed for platelet concentrate storage at room temperature for up to seven days (depending upon the type of storage container). However, it was recognized that the incidence of bacterial growth and subsequent transfusion reactions in the recipient increased to unacceptable levels with a seven day old platelet concentrate. Platelet concentrates may now be stored for no more than five days.
Blood bags used for platelet concentrate preparation are in themselves sterile, as are the connected satellite bags. One might believe, therefore, that it is a relatively simple matter to keep the blood preparation sterile during the manipulations needed to concentrate the platelets. However, bacteria can be introduced by at least two different means. First, if the donor is experiencing a mild bacteremia, the blood will be contaminated, regardless of the collection or storage method. Adequate donor histories and physicals will decrease but not eliminate this problem. See B. J. Grossman et al., Transfusion 31:500 (1991). A second, more pervasive source of contamination is the venepuncture. Even when xe2x80x9csterilexe2x80x9d methods of skin preparation are employed, it is extremely difficult to sterilize the crypts around the sweat glands and hair follicles. During venepuncture, this contaminated skin is often cut out in a small xe2x80x9ccorexe2x80x9d by a sharp needle. This core can serve to xe2x80x9cseedxe2x80x9d the blood bag with bacteria that may grow and become a risk to the recipient.
Indeed, many patients requiring platelet transfusions lack host-defense mechanisms for normal clearing and destruction of bacteria because of either chemotherapy or basic hematological disease. The growth of even seemingly innocuous organisms in stored platelets can, upon transfusion, result in recipient reaction and death. See e.g. B. A. Myhre JAMA 244:1333 (1980). J. M. Heal et al. Transfusion 27:2 (1987).
The reports assessing the extent of contamination in platelets have differed in their methods, sample size, and bacterial detection schemes. D. H. Buchholz, et al., Transfusion 13:268 (1973) reported an overall level of platelet contamination of 2.4% when a large ( greater than 1000 bags) sample was examined and extensive measures were taken for bacterial culturing. While some units were heavily contaminated after just 24 hours of storage, the incidence as a whole varied according to the age of the concentrate and increased with the widespread practice of pooling individual units; over 30% of pools were contaminated at 3 days. See also D. H. Buccholz, et al., New Eng. J. Med. 285:429 (1971). While other clinicians suggest lower numbers, recent studies indicate that septic platelet transfusions are significantly underreported. See e.g. J. F. Morrow et al. JAMA 266:555 (1991).
Pre-culturing platelets is not a solution to the bacterial contamination problem. The culture assay takes 48 hours to detect growth. Holding platelet units for an additional two days to await the results of the assay would create, ironically, a smaller margin of safety. See Table 2 in J. F. Morrow et al. JAMA 266:555 (1991). While heavily contaminated units would be detected at the outset, lightly contaminated units would be allowed to grow for two days. Older and potentially more contaminated units would end up being transfused.
Washing the blood cells (e.g. with saline) or filtering the bacteria are also not practical solutions. These techniques are time consuming and inefficient, as they can reduce the number of viable blood cells available for transfusion. Most importantly, they typically involve an xe2x80x9centryxe2x80x9d into the storage system. Once an entry is made in a previously closed system, the system is considered xe2x80x9copened,xe2x80x9d and transfusion must occur quickly, regardless of the manner in which the blood was collected and processed in the first place.
Nor are antibiotics a reasonable solution. Contamination occurs from a wide spectrum of organisms. Antibiotics would be needed to cover this spectrum. Many recipients are allergic to antibiotics. In addition, there is an every increasing array of drug-resistant strains of bacteria that would not be inactivated.
There has been interest recently in inactivation of pathogens in blood using photoreactive compounds, such as psoralens. Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985). Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058 (1977). S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982). J. Tessman et al., Biochem. 24:1669 (1985). Hearst et al., U.S. Pat. Nos. 4,124,589, 4,169,204, and 4,196,281, hereby incorporated by reference.
Psoralens have been shown to inactivate viruses in some blood products. See H. J. Alter et al., The Lancet (ii:1446) (1988). L. Lin et al., Blood 74:517 (1989). G. P. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, hereby incorporated by reference, describe the use of a combination of 8-methoxypsoralen (8-MOP) and irradiation. They show that 300 ug/ml of 8-MOP together with one hour or more of irradiation with ultraviolet light can effectively inactivate viruses. However, these treatment conditions cause harm to the blood product because of energy transfer. Their approach is only feasible if the damage to cells is specifically suppressed by limiting the concentration of molecular oxygen, a difficult and expensive process.
Isopsoralens, like psoralens, are tricyclic compounds formed by the fusion of a furan ring with a coumarin. See Baccichetti et al., U.S. Pat. No. 4,312,883. F. Bordin et al., Experientia 35:1567 (1979). F. Dall""Acqua et al., Medeline Biologie Envir. 9:303 (1981). S. Caffieri et al., Medecine Biologie Envir. 11:386 (1983). F. Dall""Acqua et al., Photochem Photobio. 37:373 (1983). G. Guiotto et al., Eur. J. Med. Chem-Chim. Ther. 16:489 (1981). F. Dall""Acqua et al., J. Med. Chem. 24:178 (1984). Unlike psoralens, the rings of isopsoralen are not linearly annulated. While able to intercalate between the base pairs of double-stranded nucleic acids and form covalent adducts to nucleic acid bases upon absorption of longwave ultraviolet light, isopsoralens, due to their angular geometry, normally cannot form crosslinks with DNA. See generally, G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985).
There are devices presently employed which emit ultraviolet radiation for activating psoralens and other photoactivated compounds. U.S. Pat. No. 5,184,020, to Hearst, et al., discloses such a device for photoactivating psoralens. However, the disclosed device is structured for the irradiation of samples in tube like vessels. It does not disclose a device for use on blood bags. Further, although the patent discloses a cooling system for the irradiated samples, this system would not work for blood bags because it depends on the circulation of fluid around the sample vessels.
Other devices are not appropriate for activating psoralens, but can be used for other purposes with blood bags. For example, U.S. Pat. Nos. 4,726,949 and 4,866,282, to Miripol, disclose such an irradiation device for use in preventing alloimmunization. This device is not practical for use in laboratories which will process large quantities of blood for sterilization. The device only supports one blood container, which would bottleneck the processing of blood. (See FIG. 1, ref. no. 10, of either Miripol patent). Further, it provides radiation of wavelength from 280 to 320 nanometers, including the 313 band, (see claim 1 of the ""282 patent) at which nucleic acids absorb radiation and could be damaged. The UVB range can also destroy platelet function. The Miripol patents state that UV-A range sources xe2x80x9cdo not provide good reduction of the lymphocyte alloimmunization effect.xe2x80x9d Column 2, line 61-64, of ""949. Finally, the Miripol patents disclose the use of only one means for cooling the system during irradiation, an exhaust fan. The goal in those patents is to maintain the heat at 31 degrees C. or less. Column 3, line 44-46. However, platelets are currently stored at 22-24 degrees C. G. Stack and L. Snyder, xe2x80x9cStorage of Platelet Concentrate,xe2x80x9d Blood Separation and Platelet Fractionation, pp. 9-125 (1991 Wiley-Liss, Inc.)
Last, there are devices disclosed which would neither be appropriate for activating psoralens nor for other uses on blood products. U.S. Pat. No. 4,421,987, to Herold, discloses an apparatus for irradiating dental objects which employs radiation in the spectral range of 400 to 500 nm, for bleaching treatment of dental parts. The device is fitted with a selective reflector which reflects from the total radiation emitted by the lamp only the spectral portion lying in the desired spectral range (approximately 400 to 500 nm) while transmitting or passing the portion of the radiation lying outside this desired spectral range. The device also has a temperature control system, employing the combination of a blower with an absorption filter which, like the reflector, removes radiation outside of the desired spectral range. This apparatus is not suited for the present purpose of a photodecontamination treatment, because it is designed for use with wavelengths of light which are damaging to some blood components, while it removes wavelengths necessary to activate certain photoreactive compounds. Further, it is not equipped with a temperature maintaining system which would keep the temperature of blood samples low enough to prevent damage.
In sum, there is a need for a means of inactivating bacteria in blood components prior to storage and transfusion in a way that lends itself to use in a closed system, such as a system of blood bags. This approach must be able to handle a high volume of blood and a variety of organisms while efficiently controlling the temperature and avoiding harm to the blood product or the transfusion recipient.
The present invention relates to a device and method for photoactivating new and known compounds. The present invention further contemplates devices for binding new and known compounds to nucleic acid. Specifically, the present invention contemplates a device for photoactivating new and known compounds so that they bind to and inactivate bloodborne pathogens. In accordance with the present invention, a nucleic acid binding compound is selectively employed to treat contamination by microorganisms.
In one embodiment, the present invention contemplates: a photoactivation device for inactivating pathogens in blood products, comprising: a housing; means for providing electromagnetic radiation to cause activation of at least one photoreactive compound, contained within said housing; means for supporting a plurality of blood bags, containing said photoreactive compound, at a fixed distance from said radiation providing means during said activation, comprising a lower ultraviolet light transparent plate assembly within said housing, upon which said blood bags can rest; and an upper ultraviolet light transparent plate assembly, positioned above said lower plate assembly, said upper and lower plate assemblies defining a channel, closed off from significant exchange with air originating from outside said housing during irradiation, in which air can be circulated to cool said blood bags. In another embodiment, said lower plate assembly comprises a top and a bottom plate and an air circulation chamber between said top and bottom plates, open to said channel to allow air exchange between said air circulation chamber and said channel.
In a preferred embodiment, the device further comprises: first temperature maintaining means, comprising: means for blowing air from outside, through said housing, between said irradiation providing means and said plate assemblies, positioned within and adjacent to said housing, for cooling said irradiation providing means; and second temperature maintaining means, positioned within said housing, for circulating cooled air through said air circulation chamber and said channel, comprising: a heat exchanger, between said plate assemblies, for absorbing heat from air present in said housing, and; means for circulating air, positioned in a fixed relationship to said heat exchanger. In one embodiment, said heat exchanger comprises a conduit having an inlet port and an outlet port so that temperature control liquid may enter and exit. In a preferred embodiment, said upper and lower plate assemblies are separated by between approximately 1 and 10 cm. However, it is preferred that when said blood bags rest upon said lower plate assembly, said upper plate assembly does not contact said blood bags.
Because of the benefits of rapid processing, in one contemplated embodiment said lower plate assembly is of dimensions sufficient to support six of said blood bags. Said blood bag supporting means may further comprise means to position a plurality of attachments connected to said blood bags, so that said attachments do not significantly reduce the intensity of radiation to said blood bags, including tubing for transferring a blood product into or out of said blood bags and a blood product storage bag. The device may further comprise means for shaking said blood bag supporting means, positioned adjacent to said blood bag supporting means, for providing mixing of a sample in a blood bag during irradiation. The present invention contemplates that said lower plate assembly has a ridged upper surface to maintain the position of said blood bags during shaking.
In a preferred embodiment, said housing comprises material which blocks said electromagnetic radiation so that users are shielded from said electromagnetic radiation during said activation. Also contemplated is a means for controlling said radiation providing means, which may comprise a plurality of detectors, positioned around said radiation providing means, to measure said electromagnetic radiation; and a feedback control, connected to said detectors, which shuts off said radiation providing means at a desired output of radiation detected by said detectors. Preferably, the intensity of radiation provided by said radiation providing means is at least 15 mW/cm2, and said radiation providing means has a high end wavelength cutoff above 400 nanometers. Additionally, it is contemplated that the upper and lower plate assemblies are comprised of material which filters said electromagnetic radiation to provide a low end wavelength cutoff below 320 nanometers.
The radiation providing means may further comprise a top bank and a bottom bank of light sources, said top bank being located above said upper plate assembly, and said bottom bank being located below said lower plate assembly. Reflecting means, adjacent to said top bank and said bottom bank of light sources, are also contemplated, which reflect electromagnetic radiation from said light sources toward said blood bag supporting means.
In an alternative embodiment of the present invention, a photoactivation device is contemplated for treating photoreactive compounds, comprising: an opaque housing; means for providing electromagnetic radiation to cause activation of at least one photoreactive compound, within said housing; means for supporting a plurality of blood bags at a fixed distance from said radiation providing means during said activation, comprising a lower ultraviolet light transparent plate assembly, within said housing, upon which said blood bags can rest; an upper ultraviolet light transparent plate assembly, positioned above said lower plate assembly, said upper and lower plate assembly defining a channel, closed off from significant exchange with air originating from outside said housing during irradiation, through which air can be circulated to cool said blood bags; first temperature maintaining means, comprising: means for blowing air from outside, through said housing, between said irradiation providing means and said plate assemblies, positioned within and adjacent to said housing, for cooling said irradiation providing means; and second temperature maintaining means, positioned within said housing, for circulating cooled air through said channel, comprising: a heat exchanger, between said plate assemblies, for absorbing heat from air present in said housing; and means for circulating air, positioned in a fixed relationship to said heat exchanger, for circulating cooled air from said heat exchanger through said channel. In one embodiment, said heat exchanger comprises a conduit having an inlet port and an outlet port so that temperature control liquid may enter and exit. Said lower plate assembly may be comprised of a top and a bottom plate, and an air circulation chamber between said top and bottom plates. In this embodiment, said means for circulating air circulates air through said air circulation chamber.
It is contemplated that said upper and lower plate assemblies are separated by between approximately 1 and 10 cm. However, in a preferred embodiment, when said blood bags rest upon said lower plate assembly, said upper plate assembly does not contact said blood bags. Also contemplated is a lower plate assembly of dimensions sufficient to support six of said blood bags. It is contemplated that the blood bag supporting means further comprises means to position a plurality of attachments connected to said blood bags, so that said attachments do not significantly reduce the intensity of radiation to said blood bags. Some such attachments comprise tubing for transferring a blood product into or out of said blood bags and a blood product storage bag. The present invention further contemplates means for shaking said blood bag supporting means, positioned adjacent to said blood bag supporting means, for providing mixing of a sample in a blood bag during irradiation. In one embodiment, said lower plate assembly has a ridged upper surface to maintain the position of said blood bags during said shaking.
The present invention contemplates a photoactivation device comprising means for controlling said radiation providing means. The means for controlling said radiation providing means may comprise: a plurality of detectors, positioned around said radiation providing means, to measure said electromagnetic radiation; and a feedback control, connected to said detectors, which shuts off said radiation providing means at a desired output of radiation detected by said detectors. In a preferred embodiment, the intensity of radiation provided by said radiation providing means is at least 15 mW/cm2 and said radiation providing means has a high end wavelength cutoff above 400 nanometers.
In one embodiment, said plate assemblies are comprised of material which removes blood product damaging wavelengths of radiation from said electromagnetic radiation. Specifically, it is contemplated that said material filters said electromagnetic radiation to provide a low end wavelength cutoff below 320 nanometers. It is also contemplated that said radiation providing means comprises a top bank and a bottom bank of light sources, said top bank being located above said upper plate assembly, and said bottom bank being located below said lower plate assembly. Reflecting means may be positioned adjacent to said top bank and said bottom bank of light sources, which reflect electromagnetic radiation from said light sources toward said blood bag supporting means.
The present invention also contemplates a method for photoactivating photoreactive compounds, comprising: supporting a plurality of blood bags, containing one or more photoreactive compounds, at a fixed distance from a fluorescent source of electromagnetic radiation; irradiating said plurality of blood bags simultaneously with electromagnetic radiation having a wavelength cutoff at approximately 320 nm, from said fluorescent source to cause activation of at least one of said photoreactive compounds; and maintaining the temperature of said blood bags at approximately room temperature during said activation, by cooling air and circulating cooled air around said blood bags in a closed system. Preferably, the fluorescent source of electromagnetic radiation delivers an intensity of electromagnetic radiation greater than 1 mW/cm2 to said blood bags.